| Liver cancer is a very high mortality rate worldwide of malignant tumors,while China is a high incidence of liver cancer.miRNA popular in recent years,the direction of medicine and biology research,which has been widely involved in the tumor confirmed a variety of biological processes.miRNA is a Class length 18-24 nt noncoding single-stranded RNA,by binding to the target mRNA 3'UTR negatively regulate transcription,thus inhibiting expression of the target gene protein.In recent years,there were reports that miR-205 is a tumor suppressor,showed low expression in liver cancer,but the molecular mechanism remains unclear.Thus,miR-205 in liver tumor suppressor role played by the molecular mechanism study helps to clarify the process of the development of liver cancer.This study details and results are summarized as follows:1.Expression analysis and promoter methylation analysis of miR-205 in hepatoma cells.Background:Under the modified promoter methylation in the case does not change the base sequence downstream gene expression regulation.Phenomenon of miR-205 differentially expressed in liver cancer cells whether promoter methylation related explore upstream regulatory mechanisms of miR-205 and miR-205 study the role of the development of liver cancer in significance.Objective:To detect a liver cell line and three hepatoma cell lines and the expression of miR-205 in patients with hepatocellular carcinoma and adjacent normal tissues and to study the differences between promoter methylation level is relevant.Methods:Quantitative PCR were detected by fluorescence liver cell line HL-7702,hepatoma cell line HepG2,BEL-7404,the level of expression of miR-205 next to SMMC-7721,and 30 cases of hepatocellular carcinoma in patients with cancer tissue.Of which 16 cases were HL-7702,HepG2,BEL-7404 cell lines and patient tissue samples were bisulfite sequencing(Bisulfite Sequencing PCR,BSP).Use methyltransferase inhibitor 5-Aza-CdR treated HepG2 cells suppressed their methylation levels,and different concentrations of inhibitors is an impact on the expression of miR-205 in HepG2 cells.Results:Hepatocellular carcinoma tissue samples expression levels of miR-205 than in adjacent normal tissue,hepatoma cell line HepG2,BEL-7404 in miR-205 expression levels below normal liver cell line HL-7702,SMMC-7721 in the expression level of miR-205 HL-7702 no significant difference.Hepatoma cell line HepG2,BEL-7404 promoter methylation levels were 70%,73.33%,HL-7702 level of promoter methylation of 46.67%,cancer tissue specimens from patients with methylation level of 66.67%,cancer adjacent tissue methylation level of 40%.Statistics 16 patients isolated tissue methylation levels found in the higher grade of pathology specimens,the lower the degree of differentiation,the higher promoter methylation levels.5-Aza-CdR treated HepG2 cells can increase the expression of miR-205,and are higher the concentration,the higher the expression level of miR-205.Conclusion:HCC cell miR-205 gene by promoter hypermethylation and inhibit gene expression,and the higher the degree of methylation,the higher the degree of malignancy of HCC.2.Screening and Identification of miR-205 target genes.Background:Protein is the main material of life activities,miRNA through negative regulation of target mRNA and inhibit expression of the target gene protein.Angling target genes of miR-205 and identification is the first step to uncover miR-205 hepatoma cells involved in molecular pathways.Objective:Screening of target genes in liver cells and miR-205 were identified from specimens to detect the expression levels in patients.Methods:The bioinformatics analysis by hybrid PCR method to obtain candidate target genes RNF43.By dual luciferase reporter gene targeting experiments show that miR-205 binding of RNF43 mRNA 3'UTR region.Further fluorescence quantitative PCR and Western blot study the regulation of miR-205 to RNF43 in mRNA and protein levels.Immunohistochemical detection of RNF43 in patients with liver cancer and cancer tissue specimens from adjacent tissues expression levels.Results:hybrid PCR method combined with bioinformatics selected six candidates for target genes.Dual luciferase reporter gene experiments confirmed that miR-205 can inhibit the expression of integrin mRNA 3'UTR binding sites of the reporter gene RNF43.Quantitative PCR confirmed that miR-205 can inhibit the expression of RNF43 mRNA upregulation,but WB confirmed that inhibit the expression of miR-205 regulated RNF43 protein proved to play a transcription of miR-205 inhibition characteristics.Immunohistochemistry results show that patients with liver cancer in cancer tissue specimens RNF43 expression levels higher than adjacent tissues.Conclusion:RNF43 as target genes of miR-205,and increased expression in HCC.3.Preliminary study of the regulation of liver cancer cell proliferation mechanism of miR-205/RNF43.Background:miR-205 can inhibit tumor cell proliferation and induce cell cycle arrest.Cell cycle is an important factor affecting,cell growth,the research of miR-205/RNF43 involved in cell cycle regulation helps to reveal miR-205 on liver cancer cell growth inhibition molecular mechanisms and to conduct targeted drugs for the study of molecular mechanisms It provides a theoretical basis.Objective:To explore the liver cells miR-205/RNF43 molecular mechanisms of inhibition of cell proliferation.Methods:The effect colony formation assay miR-205 and RNF43 on cell growth ability.Cell cycle analysis to detect miR-205 and RNF43 effect on the cell cycle.Quantitative PCR miR-205 and RNF43 checkpoint protein p53 cellcycle,p21 and Cyclin E mRNA expression levels of.WB to detect miR-205 and RNF43 checkpoint protein p53 cell cycle,p21 effect,Cyclin E protein levels.Joint co-immunoprecipitation(Co-IP)if there is interaction between the assayRNF43 protein and p53 protein ubiquitination WB detect whether the promotion of RNF43 protein ubiquitination of p53 protein degradation.Nude mice bearing HepG2 xenograft animal model,by intratumoral injection of miR-205 or RNF43 expression vector,study treatments affect tumor growth in nude mice.Results:The colony formation assay proved miR-205 inhibit HepG2 cell growth,RNF43 can relieve miR-205 on the growth of HepG2 cells in vitro.Cell cycle analysis showed that miR-205 can HepG2 cell cycle G1 phase arrest,RNF43 can be lifted affect miR-205 on HepG2 cells.Fluorescence quantitative PCR and WB experiments confirmed miR-205 of p53,p21 gene produces promote mRNA and protein expression in the level of expression of Cyclin E gene produces inhibition effect,however,while overexpression of RNF43 antagonize the above effect.miR-205 could inhibit HepG2 cell xenografts in nude mice tumor growth,RNF43 can reverse miR-205 transplanted tumor growth inhibition effect.Conclusion:miR-205/RNF43 by regulating p53,p21,Cyclin E is involved in cell cycle progression of tumor cells,affecting the growth of tumor cells. |
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